The present invention relates to compositions and crystals of androgen receptor ligand binding domain optionally in complex with its ligand. This invention also relates to methods of using the structure coordinates of the androgen receptor ligand binding domain/ligand complex to solve the structure of similar or homologous proteins or protein complexes. This invention also relates to methods for designing and selecting ligands that bind to the androgen receptor and methods of using such ligands.
The androgen receptor (AR) is a member of the steroid nuclear-receptor superfamily of ligand-dependent transcription factors. The binding of androgen to AR initiates the gene activation required for male sex development.
AR is an important target primarily in two drug discovery areas. In oncology drug discovery, inhibitors (antagonists or partial antagonists) of androgen receptor function are useful for treatment of anti-androgen refractory prostate cancer. In metabolic diseases drug discovery, agonists or partial agonists to the androgen receptor in muscle are useful to treat age-related diseases.
As with the other members of the steroid receptor family, AR has several functional domains including a DNA binding domain (DBD), and a 261 residue ligand-binding domain (LBD) (Mw=30,245 Da) which contains the androgen binding site, and is responsible for switching on the androgen function.
Development of synthetic ligands that specifically bind to androgen receptors has been largely guided by trial and error method of drug design despite the importance of the androgen receptor in physiological processes and medical conditions such as prostate cancer and modulation of reproductive organ modulation. Previously, new ligands specific for androgen receptors were discovered in the absence of information on the three dimensional structure of the androgen receptor with a bound ligand. Before the present invention, researchers were essentially discovering androgen receptor ligands by probing in the dark and without the ability to visualize how the amino acids of the androgen receptor held a ligand in its grasp.
Consequently, it would be advantageous to devise methods and compositions for reducing the time required to discover ligands to the androgen receptor, synthesize such compounds and administer such compounds to organisms to modulate physiological processes regulated by the androgen receptor.
The cDNA and amino acid sequences of human and rat androgen receptors have been described (Proc. Natl. Acad. Sci. U.S.A. 1988 85: 7211-7215). However, there have been no crystals reported of any androgen receptor. Thus, x-ray crystallographic analysis of such proteins has not been possible.
We have discovered the first crystal structure of the androgen receptor ligand binding domain (AR-LBD). Our understanding or the androgen receptor structure has allowed for the determination of the ligand binding site for selective androgen receptor modulators (SARMs).
The present invention provides crystals of AR-LBD and crystals of an AR-LBD bound to a ligand, i.e. an AR-LBD/AR-LBD ligand complex. Most preferably the AR-LBD ligand is dihydrotestosterone (DHT). Thus, the present invention is directed to a crystal of an AR-LBD comprising:
1) an AR-LBD and an AR-LBD ligand or
2) an AR-LBD without an AR-LBD ligand;
wherein said crystal diffracts to at least 3 angstrom resolution and has a crystal stability within 5% of its unit cell dimensions. The crystal of AR or AR-LBD preferably has at least 200 amino acid and preferably comprises amino acid sequence 672 to 917 of rat AR or the AR amino acid sequence 672 to 917 of human AR.
The present invention also provides the structure coordinates of the AR-LBD/AR-LBD ligand complex. The complete coordinates are listed in Table A.
The present invention also provides a method for determining at least a portion of the three-dimensional structure of molecules or molecular complexes which contain at least some structurally similar features to the androgen receptor ligand binding domain. It is preferred that these molecules or molecular complexes comprise at least a part of the ligand binding site defined by structure coordinates of AR-LBD amino acids V685, L700, L701, S702, S703, L704, N705, E706, L707, G708, E709, Q711, A735, I737, Q738, Y739, S740, W741, M742, G743, L744, M745, V746, F747, A748, M749, G750, R752, Y763, F764, A765, L768, F770, M780, M787,1869, L873, H874, F876, T877 and F878 according to Table A, or a mutant or homologue thereof. Since the protein sequences for rat and human AR LBD are identical, the human numbering system was used herein.
The present invention also provides a machine-readable data storage medium which comprises a data storage material encoded with machine readable data defined by the structure coordinates of an AR-LBD/AR-LBD ligand or ligand complex according to Table A or a homologue of the complex.
The present invention further provides a binding site in AR-LBD for an AR-LBD ligand as well as methods for designing or selecting AR modulators including agonists, partial agonists, antagonists, partial antagonists and/or selective androgen receptor modulators (SARMs) of AR using information about the crystal structures disclosed herein.